Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple steatosis (NAFL), over nonalcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis with end-stage disease. The hepatic molecular events underlying the development of NAFLD and transition to NASH are poorly understood. The present study aimed to determine hepatic transcriptome dynamics in patients with NAFL or NASH compared with healthy normal-weight and obese individuals. RNA sequencing and quantitative histomorphometry of liver fat, inflammation and fibrosis were performed on liver biopsies obtained from healthy normal-weight ( n = 14) and obese ( n = 12) individuals, NAFL ( n = 15) and NASH ( n = 16) patients. Normal-weight and obese subjects showed normal liver histology and comparable gene expression profiles. Liver transcriptome signatures were largely overlapping in NAFL and NASH patients, however, clearly separated from healthy normal-weight and obese controls. Most marked pathway perturbations identified in both NAFL and NASH were associated with markers of lipid metabolism, immunomodulation, extracellular matrix remodeling, and cell cycle control. Interestingly, NASH patients with positive Sonic hedgehog hepatocyte staining showed distinct transcriptome and histomorphometric changes compared with NAFL. In conclusion, application of immunohistochemical markers of hepatocyte injury may serve as a more objective tool for distinguishing NASH from NAFL, facilitating improved resolution of hepatic molecular changes associated with progression of NAFLD. NEW & NOTEWORTHY Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. NAFLD is associated with the metabolic syndrome and can progress to the more serious form, nonalcoholic steatohepatitis (NASH), and ultimately lead to irreversible liver damage. Using gold standard molecular and histological techniques, this study demonstrates that the currently used diagnostic tools are problematic for differentiating mild NAFLD from NASH and emphasizes the marked need for developing improved histological markers of NAFLD progression.

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in the Western world. NAFLD is a complex spectrum of liver diseases ranging from benign hepatic steatosis to its more aggressive necroinflammatory manifestation, nonalcoholic steatohepatitis (NASH). NASH pathogenesis is multifactorial and risk factors are almost identical to those of the metabolic syndrome. This has prompted substantial efforts to identify novel drug therapies for correcting underlying metabolic deficits, and to prevent or alleviate hepatic fibrosis in NASH. Available mouse models of NASH address different aspects of the disease, have varying clinical translatability, and, therefore, also show different utility in drug discovery.

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Author contributions: Kristiansen MNB, Veidal SS, Rigbolt KTG, Tølbøl KS and Feigh M performed the experiments and analyzed the data; Rigbolt KTG performed the molecular investigations; Kristiansen MNB and Veidal SS performed the histological analysis; Veidal SS, Rigbolt KTG, Roth JD, Jelsing J, Vrang N and Feigh M designed and coordinated the research; Kristiansen MNB, Veidal SS, Rigbolt KTG, Tølbøl KS, Roth JD, Jelsing J, Vrang N and Feigh M wrote the paper.Institutional review board statement: This study includes no data or material from patients. We confirm that all of the required permissions for this study were obtained from our local authorities as mentioned in the Institutional animal care and use committee statement.Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Danish Committee for animal research and covered by a personal license for Jacob Jelsing (2013-15-2934-00784). All of the institutional and national guidelines for the care and use of laboratory animals were followed.Conflict-of-interest statement: There are no patents, products in development or marked products to declare. Abstract AIM: To characterize development of diet-induced nonalcoholic steatohepatitis (NASH) by performing liver biopsy in wild-type and genetically obese mice. METHODS:Male wild-type C57BL/6J (C57) mice (DIO-NASH) and male Lep ob /Lep ob (ob /ob ) mice (ob /ob -NASH) were maintained on a diet high in trans-fat (40%), fructose (22%) and cholesterol (2%) for 26 and 12 wk, respectively. A normal chow diet served as control in C57 mice (lean chow) and ob /ob mice (ob /ob chow).After the diet-induction period, mice were liver biopsied and a blinded histological assessment of steatosis and fibrosis was conducted. Mice were then stratified into groups counterbalanced for steatosis score and fibrosis stage and continued on diet and to receive daily PO dosing of vehicle for 8 wk. Global gene expression in liver tissue was assessed by RNA sequencing and bioinformatics. Metabolic parameters, plasma liver enzymes and lipids (total cholesterol, triglycerides) as well as hepatic lipids and collagen content were measured by biochemical analysis. Non-alcoholic fatty liver disease activity score (NAS) (steatosis/inflammation/ballooning Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy Basic Study ORIGINAL ARTICLEdegeneration) and fibrosis were scored. Steatosis and fibrosis were also quantified using percent fractional area. RESULTS: Diet-induction for 26 and 12 wk in DIO-NASH and ob /ob -NASH mice, respectively, elicited progressive metabolic perturbations characterized by increased adiposity, total cholesterol and elevated plasma liver enzymes. The diet also induced clear histological features of NASH including hepatosteatosis and fibrosis. Overall, the metabolic NASH phenotype was more pronounced in ob /ob -NASH vs DIO-NASH mice. During the eight week repeated vehicle dosing period, the metabolic phenotype was sustai...

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.